Synthesis and characterization of folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29 cells

Folate–chitosan (FA–CS) conjugates synthesized by coupling FA with CS render new and improved functions because the original properties of CS are maintained and the targeting ligand of FA is incorporated. In this work, FA–CS conjugates were synthesized based on chemical linking of carboxylic group of FA with amino group of CS as confirmed by Fourier transform spectroscopy (FTIR) and nuclear magnetic resonance (¹H NMR). FA–CS conjugates displayed less crystal nature when compared to CS. The FA–CS nanoparticles (NPs) were prepared by crosslinking FA–CS conjugates with sodium tripolyphosphate (STPP). Positively charged FA–CS nanoparticles were spherical in shape with a particle size of about 100 nm. Cellular uptake of CS or FA–CS nanoparticles was assayed by fluorescent microscopy using calcein as fluorescent marker in colon cancer cells (HT-29). The FA–CS nanoparticles exhibited improved uptake of HT-29 and could become a potential targeted drug delivery system for colorectal cancer.     

Synthesis of folic acid functionalized terbium‐doped dendritic fibrous nano‐silica and Interaction with HEK 293 normal,MDA breast cancer and HT 29 colon cancer cells

A novel folic acid functionalized terbium‐doped dendritic fibrous nanoparticle (Tb@KCC‐1‐NH₂‐FA) with high surface area was synthesized using a novel hydrothermal protocol. In the present work, we report the fluorescent Tb‐doted nanomaterial with emission wavelength at 497 nm which confirms the formation of Tb@KCC‐1‐NH₂‐FA. Synthesized nanoparticles were investigated through transmission electron microscope, field emission scanning electron Microscopy, Fourier transform infrared spectra, Brunauer‐Emmett‐Teller, energy dispersive X‐ray, Zeta potential and particle size distribution values and AFM (Atomic force microscopy) techniques. Specially, our desired nanomaterial which has FA moieties on the surface of Tb@KCC‐1‐NH2‐FA where interact with folate receptor (FR) which there is on the surface of the various cancer cells. For this purpose, fluorescence microscopy images were used to prove the uptake of FA based nanomaterial with FR‐positive MDA breast cancer and HT 29 colon cancer cells. Also HEK 293 normal cells as FR‐negative cells verified the specificity of our desired nanomaterial toward the FR‐positive cells. The cytotoxicity survey of Tb@KCC‐1‐NH₂‐FA was examined by MTT assays against MDA breast cancer, HT 29 colon cancer and HEK 293 Normal cell lines which confirmed their biocompatible nature with any significant cytotoxic effects even for concentration higher than 900 μg/mL which could be used as a non‐toxic catalyst or carrier in biological ambient. Hence, Tb@KCC‐1‐NH₂‐FA were synthesized using green and hydrothermal method; the process was simple with good productivity and desired nanocomposite was non‐toxic.     

Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate

Fluorescence optical imaging technologies are currently being developed to image specific molecular targets in vivo. Detection technologies range from those providing microscopic detail to whole body imaging systems with potential clinical use. A number of target-specific near-infrared imaging probes have recently been developed to image receptors, antigens, and enzymes. The goal of the current study was to evaluate a new near-infrared (NIR) folate receptor (FR)-targeted imaging probe for its ability to improve detection of FR-positive cancers. We hypothesized that modification of folate would retain receptor affinity in vivo, despite the bulkier NIR fluorochrome, NIR2 (em = 682 nm). Cellular uptake of the NIR conjugates was significantly higher in FR-positive nasopharyngeal epidermoid carcinoma, KB cells, compared to FR-negative human fibrosarcoma, HT1080 cells. When tumors were implanted in vivo, equal-sized KB tumors showed a 2.4-fold higher signal intensity compared to HT1080 tumors (24 h). The maximum signal-to-background ratio (3-fold) was observed at 24 h in KB tumor. Injection of the unmodified NIR2 fluorochrome did not result in persistent contrast increases under similar conditions. Furthermore, tumor enhancement with the NIR2-folate probe persisted over 48 h and was inhibitable in vivo by administration of unlabeled folate. These results indicate that folate-modified NIR fluorochrome conjugate can be used for improved detection of FR-positive tumors.     

Bifunctional PAMAM dendrimer conjugates of folic acid and methotrexate with defined ratio

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.     

Folate/NIR 797-Conjugated Albumin Magnetic Nanospheres: Synthesis,Characterisation, and In Vitro and In Vivo Targeting Evaluation

A practical and effective strategy for synthesis of Folate-NIR 797-conjugated Magnetic Albumin Nanospheres (FA-NIR 797-MAN) was developed. For this strategy, Magnetic Albumin Nanospheres (MAN), composed of superparamagnetic iron oxide nanoparticles (SPIONs) and bovine serum albumin (BSA), were covalently conjugated with folic acid (FA) ligands to enhance the targeting capability of the particles to folate receptor (FR) over-expressing tumours. Subsequently, a near-infrared (NIR) fluorescent dye NIR 797 was conjugated with FA-conjugated MAN for in vivo fluorescence imaging. The FA-NIR 797-MAN exhibited low toxicity to a human nasopharyngeal epidermal carcinoma cell line (KB cells). Additionally, in vitro and in vivo evaluation of the dynamic behaviour and targeting ability of FA-NIR 797-MAN to KB tumours validated the highly selective affinity of FA-NIR 797-MAN for FR-positive tumours. In summary, the FA-NIR 797-MAN prepared here exhibited great potential for tumour imaging, since the near-infrared fluorescence contrast agents target cells via FR-mediated endocytosis. The high fluorescence intensity together with the targeting effect makes FA-NIR 797-MAN a promising candidate for imaging, monitoring, and early diagnosis of cancer at the molecular and cellular levels.     

Receptor-targeted liposomal delivery of boron-containing cholesterol mimics for boron neutron capture therapy (BNCT)

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.     

Relationships between carbon allocation and partitioning of soil respiration across world mature forests

Partitioning of soil CO₂ flux (FS) into autotrophic and heterotrophic components depends on how the plant carbon is allocated above- vs. belowground and how the belowground carbon is allocated for respiration and production of roots and their microbial associations. Data of litterfall (FA), root respiration (FR), and FS of world old-growth or mature forests (≥45 ages) were compiled, and the relationship between carbon allocation above- vs. belowground (indexed as the FA/FS ratio) and FS partitioning (indexed as the FR/FS ratio) was examined. The FA/FS ratio ranged from 0.08 to 0.64 and was positively correlated with mean annual air temperature and mean annual precipitation. The ratio increased from boreal to temperate to tropical forests, and was higher in broadleaved forests than in coniferous forests. Site-specific belowground carbon use efficiency (BCUE, root production per unit carbon used by roots and microbial associations) varied from 0.10 to 0.87, contrasting with the common assumption of a constant BCUE. Site-specific FR/FS ranged from 0.09 to 0.71 and increased with FS due to a decrease in BCUE. Deciduousness had a significant effect on the FR/FS ratios, with FR/FS ratios greater in deciduous forests than in evergreen forests. Methods of separating root respiration from soil heterotrophic respiration had a significant effect on estimated FR/FS. The estimated FR/FS ratio was negatively related to the FA/FS ratio, indicating that factors favouring carbon allocation belowground over aboveground will increase the autotrophic contribution to total soil respiration. The relatively low explaining power (r ² = 0.270) of this relationship resulted from deviations from assumptions of constant BCUE and a near steady-state belowground pools.     

Investigating Fluorescence Quenching of ZnS Quantum Dots by Silver Nanoparticles

Water dispersible zinc sulfide quantum dots (ZnS QDs) with an average diameter of 2.9 nm were synthesized in an environment friendly method using chitosan as stabilizing agent. These nanocrystals displayed characteristic absorption and emission spectra having an absorbance edge at 300 nm and emission maxima (λ _emission) at 427 nm. Citrate-capped silver nanoparticles (Ag NPs) of ca. 37-nm diameter were prepared by modified Turkevich process. The fluorescence of ZnS QDs was significantly quenched in presence of Ag NPs in a concentration-dependent manner with K _sv value of 9 × 10⁹ M⁻¹. The quenching mechanism was analyzed using Stern–Volmer plot which indicated mixed nature of quenching. Static mechanism was evident from the formation of electrostatic complex between positively charged ZnS QDs and negatively charged Ag NPs as confirmed by absorbance study. Due to excellent overlap between ZnS QDs emission and surface plasmon resonance band of Ag NPs, the role of energy transfer process as an additional quenching mechanism was investigated by time-resolved fluorescence measurements. Time-correlated single-photon counting study demonstrated decrease in average lifetime of ZnS QDs fluorescence in presence of Ag NPs. The corresponding F?rster distance for the present QD–NP pair was calculated to be 18.4 nm.     

Microarray analysis of immediate-type allergy in KU812 cells in response to fulvic acid

Fulvic acid (FA) is class of compounds of humic substances formed through the degradation of organic substances by chemical and biological processes. FA has been utilized in traditional Chinese medicine and possesses various pharmacological properties. Previously, we reported that FA extracted from solubilized excess sludge (SS-FA) had an inhibitory effect on β-hexosaminidase release in human leukemia basophilic (KU812) cells. In this study, we investigated the effects of SS-FA on the immediate-type allergic reaction and studied its possible mechanisms of action in KU812 cells following activation with phorbol myristate acetate (20 nmol L⁻¹) plus calcium ionophore A23187 (1 μmol L⁻¹) (PMACI). The inhibitory effect of SS-FA on degranulation in PMACI-stimulated KU812 cells was examined using histamine release assay. SS-FA significantly decreased the histamine release in KU812 cells at concentrations of 0.1–10.0 μg mL⁻¹. To gain more information regarding the mechanism of the suppression of degranulation following SS-FA treatment, microarray was conducted to determine which genes were differentially expressed in response to SS-FA in PMACI-activated KU812 cells. From a total of 201 genes in the DNA chip, 28 genes were up-regulated and 173 genes were down-regulated in cells pretreated with SS-FA for 15 min and stimulated with PMACI. From the 71 genes that showed more than two fold change in expression, 16 genes were significantly down-regulated that were subjected to hierarchical clustering. SS-FA affected the expression of genes that were involved in the following pathways: signal transduction, cytokine–cytokine receptor interaction, immune response, cell adhesion molecules and IgE receptor β subunit response.     

Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation

Curcumin (CUR), a plant-derived compound, exhibits versatile antitumor effects. However, its poor hydrophilic property limits its application. To circumvent these drawbacks, we encapsulated CUR in liposomes modified with folic acid for better solubility and enhanced tumor targeting. This novel formulation was prepared by a film-dispersion method and characterized by size, zeta potential, drug-loading efficiency, and physical-condition stability. In vitro, cellular uptake efficiency, cytotoxicity, and apoptosis analysis by flow cytometry were performed to evaluate tumor targeting and killing ability. Results showed that the folate-receptor (FR)-targeted liposomal CUR (F-CUR-L) performed with improved solubility, sufficient stability, and enhanced antitumor activity. Mean diameter, zeta potential, and drug-loading efficiency were 182?nm, ?26 mV, and 68%, respectively, and this formulation exhibited stability in storage at 4°C for 1 month. In vitro, FR-positive cells endocytosed more F-CUR-L than nontargeted liposomal CUR (CUR-L); thus, the former induced more cellular proliferation inhibition and higher apoptosis than the latter, and the enhanced targeting could be hindered by 1?mM of free folic acid. Further, KB cells were more sensitive to F-CUR-L, compared to Hela cells. Finally, the two kinds of tumor cells treated with F-CUR-L also showed dose- and time-dependent apoptosis.     

Salicylic acid-mediated alleviation of cadmium toxicity in hemp plants in relation to cadmium uptake,photosynthesis, and antioxidant enzymes

To assess the role of salicylic acid (SA) in alleviating cadmium (Cd) toxicity in hemp (Cannabis sativa L.) plants, the growth parameters, Cd accumulation, photosynthetic performance and activities of major antioxidant enzymes were investigated in hemp seedlings treated with 500 μM SA, under 0, 25, 50, and 100 mg Cd kg⁻¹ sands (DW) conditions, respectively. Cd exposure resulted in a small reduction in biomass (12.0–26.9% for root, and 8.7–29.4% for shoot, respectively), indicating hemp plants have innate capacity to tolerant Cd stress. This was illustrated by little inhibition in photosynthetic performance, unchanged malondialdehyde content, and enhancement of superoxide dismutase (SOD) and peroxidases (POD) activities in hemp plants. Cd content in root is 25.0–29.5 times’ higher than that in shoot, suggesting the plant can be classified as a Cd excluder. It is concluded that SA pretreatment counteracted the Cd-induced inhibition in plant growth. The beneficial effects of SA in alleviating Cd toxicity can be attributed to the SA-induced reduction of Cd uptake, improvement of photosynthetic capacity, and enhancement of SOD and POD activities.     

Adenovirus vector production using low-multiplicity infection of 293 cells

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001–0.1 transductional unit (TU) cell⁻¹. The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell⁻¹), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 × 10⁵ cells cm⁻³ were infected with Ad EGFP at a low MOI of 0.001 TU cell⁻¹, the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell⁻¹) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.     

Size-related dietary changes observed in young-of-the-year pumpkinseed (<Emphasis Type="Italic">Lepomis gibbosus</Emphasis>): stomach contents and fatty acid analyses

Changes in dietary composition and its effects on the fatty acid (FA) composition of young-of-the-year (YOY) pumpkinseed (Lepomis gibbosus) from an artificial reservoir (Mirgenbach—northeastern France) were related to body size as shown by stomach content and FA analyses. Comparisons were made between three size classes of fishes: 25–35 mm total length (TL), 35–45 mm TL and 45–55 mm TL. Diets of the youngest L. gibbosus (TL ≤ 35 mm) consisted mainly of zooplanktonic microcrustaceans and Chironomidae. Ontogenetic development influenced the FA composition of L. gibbosus. Older YOY L. gibbosus showed an increase in proportions of monounsaturated FA proportions and a decrease in polyunsaturated FA and mainly essential FA (particularly docosahexaenoic acid). The low ω3/ω6 ratio and low PUFA content (mainly on DHA) suggested that L. gibbosus would not transfer the benefits of consuming ω3 PUFA up the food web.     

Synthesis and biological evaluation of a folate-targeted rhaponticin conjugate

To improve the therapeutic effect of rhaponticin (RHA), a folate receptor (FR) targeted RHA conjugate was synthesized by utilizing a hydrophilic peptide spacer linked to folic acid (FA) via a releasable disulfide linker. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Treatment of FRHA with a reducing agent indicated that the amino-reactive derivative of RHA would be released spontaneously following disulfide bond reduction within the endosomes. FRHA also proved to be active predominantly specific against FR-positive syngeneic and xenograft models in vivo, and possible curative activity resulted with minimal to moderate toxicity. The FRHA conjugate greatly enhanced the therapeutic effects and reduced the toxicity of RHA. In conclusion, FRHA represents a folate-targeted chemotherapeutic that can produce potent activity against established sc tumors. Hence, this report has a great significance in pharmacology and clinical medicine as well as methodology.     

Comparison of tetrahydrofuran,fetal calf serum,and Tween 40 for the delivery of astaxanthin and canthaxanthin to HepG2 cells

The present investigation aimed to compare fetal calf serum (FCS) and Tween 40 with the commonly employed tetrahydrofuran (THF) with respect to cytotoxicity, stability of the solubilized carotenoids, and uptake and accumulation of the xanthophylls astaxanthin (AX) and canthaxanthin (CX) in cultured human liver cells (HepG2). Incubation of HepG2 cells for 24 h with THF (≥1.25%) or FCS (≥11.25%) with or without AX (≥25 μmol/L) or CX (≥25 μmol/L) did not affect cell viability. Tween 40 (0.25–1.25% in medium) reduced cell viability by 75–99%. The stabilities of AX and CX in cell-free RPMI 1640 medium for ≤24 h were higher when delivered with THF instead of FCS. The dose- and time-dependent accumulations of AX and CX (1–10 μmol/L) in HepG2 cells were higher when carotenoids were delivered with FCS compared to THF. In conclusion, FCS and THF, but not Tween 40, were suitable solvent systems for the delivery of AX and CX to HepG2 cells. In our experiments FCS was superior with regard to the uptake and accumulation of both carotenoids.     

Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation

Curcumin (CUR), a plant-derived compound, exhibits versatile antitumor effects. However, its poor hydrophilic property limits its application. To circumvent these drawbacks, we encapsulated CUR in liposomes modified with folic acid for better solubility and enhanced tumor targeting. This novel formulation was prepared by a film-dispersion method and characterized by size, zeta potential, drug-loading efficiency, and physical-condition stability. In vitro, cellular uptake efficiency, cytotoxicity, and apoptosis analysis by flow cytometry were performed to evaluate tumor targeting and killing ability. Results showed that the folate-receptor (FR)-targeted liposomal CUR (F-CUR-L) performed with improved solubility, sufficient stability, and enhanced antitumor activity. Mean diameter, zeta potential, and drug-loading efficiency were 182?nm, -26 mV, and 68%, respectively, and this formulation exhibited stability in storage at 4 °C for 1 month. In vitro, FR-positive cells endocytosed more F-CUR-L than nontargeted liposomal CUR (CUR-L); thus, the former induced more cellular proliferation inhibition and higher apoptosis than the latter, and the enhanced targeting could be hindered by 1?mM of free folic acid. Further, KB cells were more sensitive to F-CUR-L, compared to Hela cells. Finally, the two kinds of tumor cells treated with F-CUR-L also showed dose- and time-dependent apoptosis.     

Fluorescent cytochemistry of acid phosphatase and demonstration of fluid-phase endocytosis using an azo dye method

 The aim of this work was the development of a fluorescent microscopy technique to visualize acid phosphatase activity in living and pre-fixed cells. We have shown that a coupling azo dye method, using naphthol AS-MX phosphate (NP) as substrate and fast red TR (FR) as a diazonium salt coupling agent, gives rise to a fluorescent azo dye reaction product which permits a highly sensitive demonstration of lysosomal acid phosphatase in both living and pre-fixed monolayer cell cultures. The granular staining is prevented by inhibition of acid phosphatase activity using fluoride and/or orthovanadate in both living and pre-fixed preparations. Lysosomal staining in living cells is also abolished by inhibition of fluid-phase endocytosis using low temperatures or inhibition of oxidative phosphorylation. It was shown that whilst NP entered living cells by passive diffusion, occurrence of FR in lysosomes resulted from fluid-phase endocytosis. Spectroscopic analysis of the emission and absorption features of FR, NP, naphthol AS-MX (N), and the N–FR azo dye reaction product in solution corroborated our microscopic results. The differing uptake mechanisms, and the occurrence of lysosomally localized azo dye, were also in keeping with the predictions of quantitative structure–activity relationship models of this system. Accepted: 24 April 1997     

Effects of dietary β-glucan and rice fermented on growth performance,fatty acids,and Newcastle disease immune response in turkey broilers

Poultry production has been developing in Vietnam with challenges of disease. Thus, feed additive should be investigated not only growth but also health enhancement. Here, we aimed to determine the effects of Saccharomyces cerevisiae-fermented rice (FR) and β-glucan on turkey’s growth performance, carcass characteristics, immune and fatty acid (FA) profiles. A total of 180 turkey chicks aged 1–56 days were randomly assigned to five sextuplicate groups and the birds had ad libitum feed and water access throughout the experiment. The five treatment groups were given the same diet with different proportions of FR and β-glucan. Broilers supplemented with 4% β-glucan and 4% FR presented the highest and second-highest growth performance, respectively. The 4% β-glucan and 4% FR treatments resulted in the highest carcass characteristic values without significantly affecting the breast or thigh meat pH or cooking loss. The 4% β-glucan and 4% FR treatments maximally increased the Newcastle disease (ND) antibody titers at 28, 42 and 56 days, respectively as well as thymus organ index. The foregoing treatments did not significantly affect the blood profiles relative to the control. However, the 4% FR treatment lowered the blood cholesterol levels (p > 0.05). The total FA profiles did not significantly differ among treatments. Nevertheless, both the β-glucan and FR treatments increased the MUFA levels compared to that of the control (p > 0.05). Hence, the dietary administration of 4% β-glucan and FR to turkey broilers could effectively improve their growth performance and immunity.     

Cholesterol-modified anti-<Emphasis Type="Italic">MDR1</Emphasis> small interfering RNA: Uptake and biological activity

Small interfering RNAs (siRNA) are considered to be potential agents for specific gene silencing, but low the efficacy of siRNA delivery into cells limits their biomedical application. Accumulation of siRNA coupled with cholesterol residue at the 5′-end of the “sense” strand (chol-siRNA) was studied in HEK293, HepG2, SC1, and KB-8-5 cells. In the absence of a transfection agent, the levels of both carrier-free and chol-siRNAs were very low, whereas transfection agent substantially increased transfection rate in all cell lines; in HEK293, SC1, and KB-8-5 cells transfection efficiency of the chol-siRNA was higher than that of the corresponding unmodified siRNA. Biological activity of anti-MDR1-siRNAs targeted to the 557–577 nt region of the MDR1 gene mRNA was estimated as multiple drug resistance phenotype reverting activity of KB-8-5 cancer cells. The chol-siRNA induced cancer cells’ death in the presence of previously tolerated vinblastine doses more effectively than unmodified siRNA.     

Peptide-based pharmacomodulation of a cancer-targeted optical imaging and photodynamic therapy agent

We designed and synthesized a folate receptor-targeted, water-soluble, and pharmacomodulated photodynamic therapy (PDT) agent that selectively detects and destroys the targeted cancer cells while sparing normal tissue. This was achieved by minimizing the normal organ uptake (e.g., liver and spleen) and by discriminating between tumors with different levels of folate receptor (FR) expression. This construct (Pyro-peptide-Folate, PPF) is composed of three components: (1) pyropheophorbide a (Pyro) as an imaging and therapeutic agent, (2) peptide sequence as a stable linker and modulator improving the delivery efficiency, and (3) Folate as a homing molecule targeting FR-expressing cancer cells. We observed an enhanced accumulation of PPF in KB cancer cells (FR+) compared to HT 1080 cancer cells (FR-), resulting in a more effective post-PDT killing of KB cells over HT 1080 or normal CHO cells. The accumulation of PPF in KB cells can be up to 70% inhibited by an excess of free folic acid. The effect of Folate on preferential accumulation of PPF in KB tumors (KB vs HT 1080 tumors 2.5:1) was also confirmed in vivo. In contrast to that, no significant difference between the KB and HT 1080 tumor was observed in case of the untargeted probe (Pyro-peptide, PP), eliminating the potential influence of Pyro's own nonspecific affinity to cancer cells. More importantly, we found that incorporating a short peptide sequence considerably improved the delivery efficiency of the probe--a process we attributed to a possible peptide-based pharmacomodulation--as was demonstrated by a 50-fold reduction in PPF accumulation in liver and spleen when compared to a peptide-lacking probe (Pyro-K-Folate, PKF). This approach could potentially be generalized to improve the delivery efficiency of other targeted molecular imaging and photodynamic therapy agents.